Antimicrobial compositions

ABSTRACT

The present invention provides an antimicrobial composition comprising an antimicrobial effective amount (such as a preservative, bactericidal, and/or fungicidal effective amount) of a mixture comprising at least two of:  
     (a) lemon grass oil;  
     (b) cinnamaldehyde, cinnamon oil, cinnamomum cassia, cinnamon extract, cassia leaf oil, 3,4-dihydroxycinnamic acid or salt thereof, or a mixture thereof;  
     (c) sorbic acid, or a salt thereof;  
     (d) erythorbic acid, or a salt thereof;  
     (e) benzoic acid, or a salt thereof;  
     (f) arabinogalactan, galactoarabinan, or a mixture thereof;  
     (g) a hexahydro-iso-alpha-acid, tetrahydro-iso-alpha-acid, or a mixture thereof;  
     (h) Achillea fragrantissima oils, Santolina fragrantissima oils, Forssk oils, Lavender cotton oils; and  
     (i) Glucono Delta Lactone.  
     The present invention also provides a product (preferably a product other than a foodstuff, pharmaceutical, or cosmetic) comprising a preservative effective amount of cinnamaldehyde or a mixture of cinnamaldehyde and one or more alkanol-dialkyl hydantoins.

[0001] This application claims the benefit of prior U.S. ProvisionalApplication No. 60/403,004, filed Aug. 12, 2002, and prior U.S.Provisional Application No. 60/403,169, filed Aug. 12, 2002, the entirecontents both of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to (1) antimicrobial compositions,(2) methods of killing and/or inhibiting the growth or microorganisms,(3) preserving products with the same, and (4) methods of potentiatingantimicrobial compositions.

BACKGROUND OF THE INVENTION

[0003] Natural products, while often safe, are generally expensive anddo not have biocidal efficacy against a broad spectrum of organisms suchas gram negative and positive bacteria and fungi. Most natural productsare only effective against gram positive bacteria at relatively highconcentrations and are not effective against gram negative bacteria orfungi.

[0004] Cinnamaldehyde is a natural product which has been used (1) as aflavoring agent, (2) in preservative systems, and (3) to control insectand arachnid populations. See U.S. Pat. Nos. 4,525,480, 5,306,707,5,536,501, 5,676,958, and 5,839,224.

[0005] There is a continuing need for low cost and safe preservativesystems which are effective against a broad spectrum of microorganisms.

SUMMARY OF THE INVENTION

[0006] The present invention provides an antimicrobial compositioncomprising an antimicrobial effective amount (such as a preservative,bactericidal, and/or fungicidal effective amount) of a mixturecomprising at least two of:

[0007] (a) lemon grass oil;

[0008] (b) cinnamaldehyde, cinnamon oil, cinnamomum cassia, cinnamonextract, cassia leaf oil, 3,4-dihydroxycinnamic acid or salt thereof, ora mixture thereof;

[0009] (c) sorbic acid, or a salt thereof;

[0010] (d) erythorbic acid, or a salt thereof;

[0011] (e) benzoic acid, or a salt thereof;

[0012] (f) arabinogalactan, galactoarabinan, or a mixture thereof;

[0013] (g) a hexahydro-iso-alpha-acid, tetrahydro-iso-alpha-acid, or amixture thereof;

[0014] (h) Achillea fragrantissima oils, Santolina fragrantissima oils,Forssk oils, Lavender cotton oils; and

[0015] (i) glucono delta lactone.

[0016] Preferably the mixtures of the present invention include anantimicrobial (e.g., preservative, bactericidal, and/or fungicidal)synergistic effective amount of the aforementioned ingredients.

[0017] Preferred mixtures of the present invention include, but are notlimited to, those shown in the table below. Mixture No. Component (a)Component (b) Component (c) 1 cinnamaldehyde, sorbic acid — lemon grassoil, or a salt thereof arabinogalactan, galactoarabinan, or a mixturethereof 2 cinnamaldehyde achillea oil, — arabinogalactan,galactoarabinan, or a mixture thereof 3 cinnamaldehyde arabinogalactan,sorbic acid or salt galactoarabinan, thereof or a mixture thereof 4cinnamaldehyde sorbic acid — or a salt thereof 5 cinnamaldehydeerythorbic acid — or a salt thereof 6 benzoic acid or a salt erythorbicacid — thereof (e.g., or a salt thereof sodium benzoate) 7 sorbic acidor erythorbic acid — a salt thereof or a salt thereof 8 cinnamaldehyde,benzoic glucono delta — acid or a salt lactone thereof (e.g., sodiumbenzoate), or sorbic acid or a salt thereof

[0018] In all of the aforementioned mixtures containing erythorbic acid(or salt thereof) or glucono delta lactone, the erythorbic acid (or saltthereof) or glucono delta lactone potentiates the antimicrobial efficacyof the preservative (e.g., sorbic acid or benzoic acid) in the mixture.

[0019] Another embodiment is a method of killing and/or inhibiting thegrowth of microorganisms on a substrate or in or on a product byapplying an effective amount of the antimicrobial composition of thepresent invention to the substrate or the product.

[0020] Another embodiment is a method for potentiating the antimicrobialefficacy of an antimicrobial composition containing sorbic acid, benzoicacid, or salts thereof, by adding or including erythorbic acid or a saltthereof, or glucono delta lactone in the antimicrobial composition.

[0021] Yet another embodiment is a product comprising an antimicrobial,preservative, bactericidal, and/or fungicidal effective amount of theantimicrobial composition of the present invention. The product may be asolid or liquid. The antimicrobial compositions of the present inventionare particularly effective as preservatives for personal care products.

[0022] Yet another embodiment is a method of preserving a product (e.g.,a personal care product) by incorporating a preservative effectiveamount of the antimicrobial composition of the present invention intothe product.

[0023] Yet another embodiment is a method of killing and/or inhibitingthe growth of tree or other plant fungus on a plant (such as a tree) byapplying an effective amount of the antimicrobial composition of thepresent invention to the plant and/or the soil surrounding the plant.

[0024] Yet another embodiment is a product (preferably a product otherthan a foodstuff, pharmaceutical, or cosmetic) comprising a preservativeeffective amount of cinnamaldehyde or erythorbic acid or a salt thereof(e.g., sodium erythorbate). The product is generally substantially freeor completely free of parabens (such as methylparaben, ethylparaben, andpropylparaben). The product may be, for example, a household (e.g.,personal care), industrial, or institutional product. Preferred personalcare products include, but are not limited to, shampoos, lotions (e.g.,body lotions), conditioners, and soaps. Suitable household productsinclude, but are not limited to, fabric softeners, laundry detergents,and hard surface cleaners. According to one embodiment, the productcontains less than about 1, 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09,0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01% by weight ofparabens, based upon 100% total weight of product. According to onepreferred embodiment, the product contains less than a smellingeffective amount of cinnamaldehyde. The product preferably contains morethan 0.01, 0.03, 0.05, 0.07, 0.09, or 0.1% by weight of cinnamaldehyde.The product is preferably substantially free or completely free ofcinnamon oil. According to one embodiment, the product does not containa preservative effective amount of a preservative other thancinnamaldehyde or erythorbic acid or a salt thereof. According toanother embodiment, the only preservative in the product iscinnamaldehyde or erythorbic acid or a salt thereof.

[0025] Yet another embodiment is a method of killing and/or inhibitingthe growth of microorganisms in or on a product (such as a product otherthan a foodstuff, pharmaceutical, or cosmetic) comprising applying aneffective amount of cinnamaldehyde to the product. The product ispreferably substantially free or completely free of parabens.

[0026] Yet another embodiment is a method of preserving a product(preferably a product other than a foodstuff, pharmaceutical, orcosmetic) comprising applying an effective amount of cinnamaldehyde tothe product. The product may be substantially free or completely free ofparabens.

[0027] Yet another embodiment is a method of killing and/or inhibitingthe growth of microorganisms in or on a product (such as (i) a productother than a foodstuff or (ii) a product other than a foodstuff,pharmaceutical, or cosmetic) comprising applying an effective amount oferythorbic acid or a salt thereof to the product.

[0028] Yet another embodiment is a method of preserving a product(preferably (i) a product other than a foodstuff or (ii) a product otherthan a foodstuff, pharmaceutical, or cosmetic) comprising applying aneffective amount of erythorbic acid or a salt thereof to the product.The product may be substantially free or completely free of parabens.

[0029] Yet another embodiment of the present invention is a method ofkilling and/or for inhibiting the growth of microorganisms on asubstrate by applying an antimicrobial or preservative effective amountof cinnamaldehyde or erythorbic acid or a salt thereof (preferablywithout applying any parabens).

[0030] Yet another embodiment is a preservative formulation comprisingan antimicrobial synergistic mixture comprising cinnamaldehyde and atleast one conventional personal care preservative, such asisothiazolinones, benzisothiazolinones, and/or formaldehyde donors, suchas alkanol substituted dialkylhydantoins. Preferably, the alkanolsubstituted dialkyl hydantoin is a compound of formula:

[0031] wherein R₁ and R₂ are each independently hydrogen or (CH₂)OH,with the proviso that both R₁ and R₂ cannot be hydrogen, and R₃ and R₄are each independently methyl, ethyl, propyl, or aryl. Preferred alkanolsubstituted dialkylhydantoins include, but are not limited to,1,3-dimethylol-5,5-dimethylhydantoin (DMDMH) and monomethyloldimethylhydantoin (MMDMH). Preferably, the preservative formulationcomprises a preservative effective amount of the synergistic mixture.According to one embodiment, the preservative formulation comprises abatericidally and/or fungicidally effective amount of the synergisticmixture. The preservative formulation may contain less than a smellingeffective amount of cinnamaldehyde. Preferably, the preservativeformulation is substantially free or completely free of parabens. Thepreservative formulation may be incorporated into a product, such asthose discussed in this application.

[0032] Yet another embodiment is a method of killing and/or inhibitingthe growth of microorganisms in or on a product (such as a product otherthan a foodstuff, pharmaceutical, or cosmetic) comprising applying aneffective amount of the aforementioned preservative formulation to theproduct. According to one embodiment, the product is substantially freeor completely free of parabens.

[0033] Yet another embodiment of the present invention is a method ofkilling and/or for inhibiting the growth of microorganisms on asubstrate by applying an antimicrobial or preserving effective amount ofthe preservative formulation of the present invention.

[0034] Yet another embodiment is a method of killing and/or inhibitingthe growth of fungi on a substrate comprising applying an effectiveamount of the aforementioned preservative formulation to the product.According to one embodiment, the product is substantially free orcompletely free of parabens.

[0035] The formulations and products of the present invention preferablyhave a pH less than 10, 9, 8.5, or 8.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 0.5% (w/w) lemon grass oil, (c) ashampoo containing 1.2% (w/w) potassium sorbate, and (d) a shampoocontaining 0.5% (w/w) lemon grass oil and 0.3% (w/w) potassium sorbateafter 21 days (based on bacterial count).

[0037]FIG. 2 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 0.05% (w/w) cinnamaldehyde, (c) ashampoo containing 1.2% (w/w) potassium sorbate, (d) a shampoocontaining 0.05% cinnamaldehyde and 0.5% potassium sorbate, and (e) ashampoo containing 0.1% (w/w) cinnamaldehyde and 0.5% (w/w) potassiumsorbate after 21 days (based on bacterial count).

[0038]FIG. 3 shows a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 0.05% (w/w) cinnamaldehyde, (c) ashampoo containing 1.0% (w/w) achillea oil, and (d) a shampoo containing0.05% (w/w) cinnamaldehyde and 0.75% (w/w) achillea oil after 21 days(based on bacterial count).

[0039]FIG. 4 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 0.1% (w/w) cinnamaldehyde, (c) ashampoo containing 1.0% (w/w) Hexahop Gold™, and (d) a shampoocontaining 0.1% (w/w) cinnamaldehyde and 0.4% (w/w) Hexahop Gold™ after7 days (based on fungal count).

[0040]FIG. 5 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 1.0% (w/w) Larex™ (arabinogalactan),(c) a shampoo containing 0.6% (w/w) potassium sorbate, and (d) a shampoocontaining 0.5% (w/w) Larex™ and 0.5% (w/w) potassium sorbate after 14days (based on fungal count).

[0041]FIG. 6 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing 0.25% (w/w) lemon grass oil, (c) ashampoo containing 0.6% (w/w) potassium sorbate, and (d) a shampoocontaining 0.1% (w/w) lemon grass oil and 0.5% (w/w) potassium sorbateafter 7 days (based on fungal count).

[0042]FIG. 7 is a bar graph of the stability of (a) an unpreservedshampoo, (b) a shampoo containing (w/w) Hexahop Gold™, (c) a shampoocontaining 0.6% (w/w) potassium sorbate shampoo, (d) a shampoocontaining 0.1% (w/w) cinnamaldehyde, and (e) a shampoo containing 0.3%(w/w) Hexahop Gold™, 0.1% (w/w) cinnamaldehyde, and 0.6% (w/w) potassiumsorbate after 7 days.

DETAILED DESCRIPTION OF THE INVENTION

[0043] Definitions

[0044] The term “microorganisms” includes, but is not limited to,bacteria, fungi, yeasts, algae, insects, and pests.

[0045] The term “about” or “approximately” means within an acceptableerror range for the particular value as determined by one of ordinaryskill in the art, which will depend in part on how the value is measuredor determined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

[0046] The term “personal care products” refers to products intended forapplication to the human body, such as to skin, hair, and nails,including, but not limited to, shampoos, conditioners, creams, lotions(such as body lotions), cosmetics, and soaps.

[0047] The term “smelling effective amount” refers to a sufficent amountof an agent incorporated into a product to give the product an odor.

[0048] The term “potentiating” refers to the ability of a compound orcomposition to enhance or increase the effect of an antimicrobialcompound or composition. Preferably, the efficacy of the combinedmixture is greater than the additive effect of the ingredients.

[0049] Suitable salts of sorbic acid, erythorbic acid, and benzoic acidinclude, but are not limited to, alkali metal or alkali earth metalsalts, such as potassium and sodium.

[0050] Components for Mixtures

[0051] Cinnamaldehyde from any source may be used in the presentinvention. For example, the cinnamaldehyde may be derived from cinnamonbark extracts (such as from bark and leaf), cassia leaf oil, cinnamomumcassia, cinnamon oils, cinnamal, cinnamyl alcohol, and mixtures thereof.

[0052] A preferred salt of sorbic acid is potassium sorbate.

[0053] A preferred salt of erythorbic acid is sodium erythorbate.

[0054] A preferred salt of benzoic acid is sodium benzoate.

[0055] Arabinogalactan and galactoarabinan may be derived from Larextrees. Arabinogalactan is available as Larex UF™ from Larex Inc. ofWhite Bear Lake, Minn.

[0056] Preferred hexahydro-iso-alpha-acids andtetrahydro-iso-alpha-acids are those obtained from hops extracts, suchas Hexahop Gold™ (also referred to as Hexahop herein) available fromJohn I. Haas, Inc. of Washington, D.C.

[0057] A preferred Achillea fragrantissima oils is Achillea oil.

[0058] According to a specific embodiment, the antimicroial compositioncontains at least 0.1% of sorbic acid, or a salt thereof, such aspotassium sorbate.

[0059] Examples of Preferred Mixtures

[0060] (i) Cinnamaldehyde and Sorbic Acid, Erythorbic Acid, or a SaltThereof

[0061] A preferred mixture is cinnamaldehyde and sorbic acid or a saltthereof, such as potassium sorbate. Another preferred mixture iscinnamaldehyde and erythorbic acid or a salt thereof, such as sodiumerythorbate. The weight ratio of cinnamaldehyde to (i) sorbic acid or asalt thereof or (ii) erythorbic acid or a salt thereof is preferablyfrom about 10:1 to about 0.1:1 and more preferably from about 5:1 toabout 0.2:1.

[0062] Concentrates of the mixture preferably include from about 2 toabout 40% by weight of cinnamaldehyde and from about 10 to about 60% byweight of sorbic acid, erythorbic acid, or a salt thereof, in water,with or without a hydroxyl co-solvent (such as glycerin or ethanol,which increase the solubility and stability of the cinnamaldehyde in theblends).

[0063] Preferably, the pH of formulations including a mixture of (i)cinnamaldehyde and (ii) sorbic acid, erythorbic, or a salt thereof isless than 10, 9, 8.5, or 8. At a pH of less than 9, such formulationsexhibit improved color stability. According to one preferred embodiment,the pH of a formulation containing a mixture of cinnamaldehyde andsorbic acid, erythorbic acid, or a salt thereof is lowered withhydrochloric acid. Preferably, a sufficient amount of hydrochloric acidis included in the formulation to lower its pH to less than 9, 8.5, or8.

[0064] A preferred preservative formulation includes from about 5 toabout 20% (w/w) cinnamaldehyde, from about 20 to 50% potassium sorbate,ethanol, and water. A more preferred preservative formulation includesabout 15% cinnamaldehyde, about 40% potassium sorbate, 10% ethanol, and35% water.

[0065] (ii) Combinations of Erythorbic Acid or a Salt Thereof, CitricAcid or a Salt Thereof, Glucono Delta Lactone, Benzoic Acid or a SaltThereof, Sorbic Acid, EDTA, or a Salt Thereof

[0066] Another preferred mixture is (a) erythorbic acid or a saltthereof (e.g., sodium erythorbate) and (b) one or more of (i) citricacid or a salt thereof, (ii) glucono delta lactone, (iii) benzoic acidor a salt thereof (e.g., sodium benzoate), (iv) sorbic acid or a saltthereof, or (v) ethylenediaminetetraacetic acid (EDTA) or a saltthereof.

[0067] Another preferred mixture is (a) benzoic acid or a salt thereof(e.g., sodium benzoate) and (b) one or more of (i) citric acid or a saltthereof, (ii) glucono delta lactone, (iii) sorbic acid or a saltthereof, or (iv) ethylenediaminetetraacetic acid (EDTA) or a saltthereof.

[0068] Erythorbic acid and salts thereof typically are not color stablein formulations, such as shampoos. Surprisingly, it has been found thatthese mixtures are color stable. It has also been surprisingly foundthat erythorbic acid and salts thereof and glucono delta lactonepotentiate the biocidal efficacy of citric acid, benzoic acid, EDTA, andsalts thereof.

[0069] Preferred mixtures include, but are not limited to, those in thetable below. Preferred and more preferred weight ratios are alsoprovided in the table. More Mixture Preferred Preferred No. Component(a) Component (b) Weight Ratio Weight Ratio 1 Benzoic Acid ErythorbicAcid about 0.1:1 about 0.2:1 or a Salt or a Salt Thereof to about 20:1to about 5:1 Thereof Thereof 2 Sorbic Acid or Erythorbic Acid about0.1:1 about 0.2:1 or a Salt or a Salt to about 20:1 to about 5:1 ThereofThereof 3 Benzoic Acid Glucono Delta about 0.1:1 about 0.2:1 or a SaltLactone to about 20:1 to about 5:1 Thereof 4 Glucono Delta ErythorbicAcid about 0.1:1 about 0.2:1 Lactone or a Salt to about 20:1 to about5:1 Thereof 5 Glucono Delta Benzoic Acid about 0.1:1 about 0.2:1 Lactoneor a Salt to about 20:1 to about 5:1 Thereof

[0070] More preferred mixtures include, but are not limited to, thoseshown in the table below. More Mixture Preferred Preferred No. Component(a) Component (b) Weight Ratio Weight Ratio 1 Sodium Sodium about 1:1 toabout 3:1 Benzoate Erythorbate about 5:1 2 Potassium Sodium about 1:1 toabout 3:1 Sorbate Erythorbate about 5:1 3 Sodium Glucono Delta about 1:1to about 3:1 Benzoate Lactone about 5:1 4 Glucono Delta Sodium about 1:1to about 3:1 Lactone Erythorbate about 5:1 5 Glucono Delta SodiumBenzoate about 1:1 to about 3:1 Lactone about 5:1

[0071] Antimicrobial Compositions

[0072] The antimicrobial compositions of the present invention areuseful as antimicrobial, fungicidal, and bactericidal agents (such asagainst allergens, tree and plant fungi, and plant and tree bacteria)and as preservatives in the papermaking, textile, agricultural, andcoating industries and in personal care, household, industrial, andinstitutional products. The antimicrobial composition may beincorporated into substrates susceptible to microbial growth to preservethem. For example, the preservative system may be incorporated into orbe a personal care product, such as a shampoo, conditioner, cream,lotion (such as body lotion), cosmetic, or a household product, such asa fabric softener, laundry detergent, or hard surface cleaner; or anindustrial product, such as paint, coatings, wood, textile, adhesive,sealant, leather, rope, paper, pulp, paper board, sheet rock, ceilingtiles, plastic, fuel, petroleum, oil, rubber working fluid, metalworking fluid, starches (such as pet food starch), or mineral slurry,such as a slurry of clay, calcium carbonate, or titanium oxide (TiO₂).

[0073] Generally, the product contains an antimicrobial, preservative,bactericidal, and/or fungicidal effective amount of the antimicrobialcomposition. According to one embodiment, the product contains fromabout 0.01 to about 2.0% by weight of each component of theantimicrobial composition, based upon 100% total weight of product.According to another embodiment, the product includes from about 0.1 toabout 1 or 2% by weight of the antimicrobial composition, based upon100% weight of total product.

[0074] Cinnamaldehyde Preservative Systems

[0075] Cinnamaldehyde and mixtures of (i) cinnamaldehyde and (ii) atleast one of an alkanol dialkyl hydantoin, isothiazolone, andbenzisothiazolinone (hereinafter referred to as “the preservativesystem”) are useful as antimicrobial, fungicidal, and bactericidalagents (such as against allergens, tree fungi, and tree bacteria) and aspreservatives in the papermaking, textile, agricultural, and coatingindustries and in personal care, household, industrial, andinstitutional products. The preservative system may be incorporated intosubstrates susceptible to microbial growth to preserve them. Forexample, the preservative system may be incorporated into or be apersonal care product, such as a shampoo, conditioner, cream, lotion(such as body lotion), cosmetic, or soap; a household product, such as afabric softener, laundry detergent, or hard surface cleaner; or anindustrial product, such as paint, coatings, wood, textile, adhesive,sealant, leather, rope, paper, pulp, paper board, sheet rock, ceilingtiles, plastic, fuel, petroleum, oil, rubber working fluid, metalworking fluid, starches (such as pet food starch), or mineral slurry,such as a slurry of clay, calcium carbonate, or titanium oxide (TiO₃).

[0076] Generally, the antimicrobial composition and preservative systemof the present invention acts quickly (e.g., reduces the microorganism(e.g., bacteria and/or fungi) count by 95, 99, 99.9, or 99.99% typicallywithin an hour) and maintains efficacy (e.g., maintains less than 10cfu/g) over long periods of time (e.g., for at least 7, 10, 14, or 28days). The term “preservative effective amount” refers to an amount ofthe preservative system which maintains the microorganism count below1000, 100, or 10 cfu/g for at least 1, 4, 7, 10, 14, or 28 days.

[0077] The antimicrobial composition and preservative system may includea solvent, such as water and water miscible solvents, including, but notlimited to, alcohols (e.g., methanol, ethanol, propanol, iso-propanol,and butanol), glycols (e.g. glycerin, diglycerin, butylene glycol,butoxydiglycol, propylene glycol, and dipropylene glycol), esters,ethers, polyethers, and any combination of any of the foregoing. Forexample, the solvent may comprise water and one or more glycol and/orone or more alcohol, such as glycerin, phenoxyethanol, benzyl alcohol,or ethanol. A specific solvent system comprises water and and a glycol,such as glycerin. A second specific solvent system comprises water andan alcohol, such as ethanol.

[0078] Other adjuvants may be included in the antimicrobial compositionand preservative system as known to one of ordinary skill in the art.Suitable adjuvants include, but are not limited to, preservatives;solubilizing agents; chelating agents, such asethylenediaminetetraacetic acid (EDTA) and salts thereof and zeolites;surfactants, such as cationic, anionic, nonionic, and amphotericsurfactants; antioxidants, such as butylated hydroxyanisole (BHA) andbutylhydroxytoluene (BHT); amine oxides; tertiary amines; zinccompounds; hydrotropes; fluoride compounds; magnesium salts; calciumsalts; carboxylic acids; phosphates; phosphonates; formaldehyde donors;glycereth-7; myristyl myristate; glutaraldehydes; biguanides; naturalproducts, such as geranoil, usnic acid, and tea tree oils; and anycombination of any of the foregoing. Suitable preservatives include, butare not limited to, quaternary ammonium chlorides; quaternary ammoniumcarbonates; benzalkonium chloride; iodine containing compounds, such as3-iodo-2-propynyl butyl carbamate (IPBC); hydantoins, such asdimethylhydantoin and halogenated hydantoins; isothiazolinones;parabens, such as methylparaben, ethylparaben, and propylparaben;dehydroacetic acid and salts thereof; isocil; chloroxylenol;chlorhexidine; phenoxyethanol; benzyl alcohol; phenethyl alcohol;benzoic acid and salts thereof such as sodium benzoate; chlorobutanol;sorbic acid and salts thereof; triclosan; triclocarban; and anycombination of any of the foregoing.

[0079] The antimicrobial composition and preservative system may beincorporated into an aqueous or oil based system or an emulsion. Asuitable solvent for an oil based system is phenoxyethanol and/or benzylalcohol.

[0080] The antimicrobial composition can be a liquid or a solid.

[0081] When the synergistic mixture contains only two ingredients fromthe list above, the weight ratio of the first component to the secondcomponent typically ranges from about 0.01:100 to about 100:0.01,preferably ranges from about 0.1:20 to about 20:0.1, and more preferablyranges from about 1:10 to about 10:1. When the synergistic mixturecontains three components, the third component can be in any amount, buttypically the weight ratio of the third component to either of the firsttwo components is from about 0.01:100 to about 100:0.01.

[0082] To prepare a formulation containing the product of the presentinvention, a concentrate of the antimicrobial composition andpreservative system is generally first prepared. The concentrate mayinclude from about 0.01 to about 100% by weight of the antimicrobialcomposition and preservative system and preferably contains from about 5to about 80% by weight of the antimicrobial composition, based upon 100%total weight of concentrate. For a two-component antimicrobialcomposition, the concentrate broadly contains from about 0.01 to about99.99% by weight of the first component and from about 99.99% to about0.01% by weight of the second component (based upon 100% total weight ofconcentrate). When the preservatives system is cinnamaldehyde, theconcentrate may include from about 0.01 to about 100% cinnamaldehyde byweight and preferably contains from about 5 to about 80% cinnamaldehydeby weight, based upon 100% total weight of concentrate. Table Aillustrates the components and the ranges of components present in atypical concentrate for the cinnamaldehyde/alkanol substituteddialkylhydantoin mixtures (based upon 100% total weight of concentrate).TABLE A Alkanol Substituted Dialkylhydantoin, Isothiazolinone, RangesCinnamaldehyde Benzisothiazolinone Broad from about 0.01 to about 99.99%from about 99.99 to about 0.01% Preferred from about 5 to about 95% fromabout 95 to about 5%

[0083] Before use, the concentrate is diluted, preferably with the samesolvent as was used in the concentrate, and/or incorporated into aproduct. Use dilutions of the composition typically comprise anantimicrobial, preservative, fungicidally, or bactericidally effectiveamount of the antimicrobial composition or preservative system.

[0084] Generally, use dilutions contain from about 0.0001% or 0.01% toabout 2% by weight of the concentrate. According to one preferredembodiment, use dilutions contain from about 0.1 to about 1% by weightof the concentrate. In more preferred embodiments, the use dilutioncontains 0.2, 0.25 or 0.30% by weight of the concentrate. The usedilution generally contains from about 0.01, to about 2.0% by weight ofeach antimicrobial ingredient, based upon 100% total weight of usedilution. According to a preferred embodiment, the antimicrobialcomposition contains from about 0.001 to about 10%, preferably fromabout 0.01 to about 1%, and more preferably from about 0.05 to about0.5% by weight of each antimicrobial ingredient (e.g., cinnamaldehyde).When the preservative system is cinnamaldehyde, the use dilution maycontain from about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09, or 0.1% to about 1, 0.5, 0.4, 0.3, 0.25, 0.2, 0.15,0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01% by weightbased upon 100% total weight of use dilution. Table B illustrates thecomponents and generally the ranges of components present in the usedilution (based upon 100% total weight of use dilution). TABLE B AlkanolSubstituted Dialkylhydantoin, Isothiazolinone, Ranges CinnamaldehydeBenzisothiazolinone Broad from about 0.001 to about from about 0.001 toabout 10% 10% Preferred from about 0.01 to about from about 0.01 toabout 1% 1% More from about 0.05 to about from about 0.05 to aboutPreferred 0.5% 0.5%

[0085] According to another embodiment, the aforementioned preservativesystem is incorporated into a product at a concentration of about 0.1 toabout 1 or 2% by weight, based upon 100% total weight of product.

[0086] Another embodiment of the present invention is a method forinhibiting the growth of microorganisms, bacteria (e.g., S. aureus (ATCC# 6538), P. aeruginosa (ATCC # 9027), and E. coli (ATCC # 8739)), and/orfungi (including plant and tree fungi) (e.g., Candida albicans,Aspergillus niger and Phytophthora ramrum) on a substrate by applying anantimicrobial, preservative, bactericidal, or fungicidal effectiveamount of the antimicrobial composition or preservative system of thepresent invention to the substrate. The antimicrobial composition orpreservative system may be applied to the substrate by any method knownin the art including, but not limited to, brushing, dipping, soaking,vacuum impregnation, and pressure treatment. A specific embodiment is amethod for inhibiting the growth of the tree fungus Phytophthora ramrumby applying a fungicidal effective amount of the antimicrobialcomposition or preservative system of the present invention to the treefungas or substrate (such as a tree) on which the tree fungas grows.Phytophthora ramrum causes Sudden Oak Death.

[0087] The antimicrobial composition of the present invention may beprepared by mixing the antimicrobial components, and optionally,solvents, and adjuvants. The mixture may be heated and/or stirred toexpedite mixing.

EXAMPLES

[0088] The following examples illustrate the invention withoutlimitation. All parts and percentages are given by weight unlessotherwise indicated.

Example 1

[0089] Each anionic shampoo sample in FIGS. 1-3 were tested as follows.A standardized mixed bacterial solution was prepared according to thefollowing procedure. 3 agar stabs of S. aureus (ATCC # 6538), P.aeruginosa (ATCC # 9027), and E. coli (ATCC # 8739) were separatelyincubated at about 35° C. for about 24 hours. Each stab was then washedwith 3 mL of sterile 0.85% saline solution. The washes of the 3 stabswere pooled together to form an organism mixture. The absorbance of theorganism mixture at 530 nm was adjusted to about 1.00 by adding saline.The spectrometer was calibrated with a saline blank. A 5 mL aliquot ofthe organism mixture was mixed together to produce the standardizedmixed bacterial solution. Then, 40 g of each shampoo sample wasinoculated with 0.2 mL of the standardized mixed bacterial solution andmixed. 1 g of the mixture was added to a sterile 20×150 mm screw captest tube.

[0090] 9 mL of sterile D/E neutralizer broth was added to the test tubeand mixed to form a 10⁻¹ dilution. Serial dilutions were preparedthrough to a 10⁻⁶ dilution with phosphate buffered water. The serialdilutions were plated onto Tryptic Soy Agar and incubated for 2 days atabout 35° C. Bacteria counts were performed after 21 days.

[0091] The anionic protein shampoo composition was comprised of 35% byweight of sodium lauryl ether sulfate; 25% by weight of triethanolaminelauryl sulfate; 3% by weight coconut diethanolamide (cocamide DEA); 1%by weight of hydrolyzed collagen, available as Polypro 5000™ from HormelFoods of Austin, Minn.; and 36% by weight of deionized water.

[0092] The antimicrobial composition containing samples were prepared bymixing the appropriate amounts of the antimicrobial ingredients and theaforementioned anionic protein shampoo composition and heating themixture to about 50° C. for about 15 minutes.

[0093] The results are shown in FIGS. 1-3.

Example 2

[0094] Each anionic shampoo sample in FIGS. 4-7 were tested as follows.A standard mixed bacterial solution was prepared according to thefollowing procedure. 2 agar slants of Candida albicans and 4 agar slantsof Aspergillus niger were separately incubated at about 25° C. for about48 hours and 7 days, respectively. Each slant was washed with 3 mL ofsterile 0.85% saline solution, collected and macerated in a tissuegrinder. Sufficient amounts of 0.85% saline solution were added to eachslant to obtain a visual count under a microscope with a NeubauerHemocytometer of each innoculum of C. albicans and A. niger. Equalvolumes of each standardized innoculum of C. albicans and A. niger weremixed together to form the standardized mixed fungal solution.

[0095] 40 g of each shampoo sample was inoculated with 0.4 mL of thestandardized mixed fungal solution and mixed. 1 g of the mixture wasadded to a sterile 20×150 mm screw cap test tube.

[0096] 9 mL of sterile D/E neutralizer broth was added to the test tubeand mixed to form a 10⁻¹ dilution. Serial dilutions were preparedthrough to a 10⁻⁶ dilution with phosphate buffered water. The serialdilutions were plated onto Sabourand dextrose agar and incubated 5 daysat about 25° C. Fungal counts were performed after 0, 7, and/or 14 days.

[0097] The anionic protein shampoo composition is described inExample 1. The shampoo samples were prepared by mixing the appropriateamounts of the antimicrobial ingredients and the anionic protein shampoocomposition and heating the mixture to about 50° C. for about 15minutes.

[0098] The results are shown in FIGS. 4-7.

Example 3

[0099] The procedure described in Example 1 was repeated with thepreservative formulations set forth in Table 1 below. The pH of theshampoo was adjusted to 6.5. The results are also shown in Table 1.TABLE 1 Day 0 Day 7 Day 14 Day 28 Preservative Formulation cfu/g. cfu/g.cfu/g. cfu/g. 0.3% w/w of a mixture 1-3 × 10⁶ <10 <10 <10 containing 75%potassium sorbate and 5% sodium erythorbate 0.3% w/w of a mixture 1-3 ×10⁶ <10 <10 <10 containing 75% sodium benzoate and 25% sodiumerythorbate 0.45% w/w sodium 1-3 × 10⁶ >3 × 10⁶ >3 × 10⁶ >3 × 10⁶erythorbate 0.45% w/w sodium 1-3 × 10⁶ 1 × 10⁵ 7 × 10⁵ <10 benzoate0.45% w/w potassium 1-3 × 10⁶ 1 × 10⁵ 6 × 10⁴ N.D. sorbate UnpreservedShampoo 1-3 × 10⁶ >3 × 10⁶ >3 × 10⁶ >3 × 10⁶

[0100] From Table 1, synergism for (1) a 0.3% dilution of potassiumsorbate (75%) and sodium erythorbate (25%) and (2) a 0.3% dilution ofsodium benzoate (75%) and sodium erythorbate (25%) against mixedbacteria in shampoo was calculated by the method described in C. E. Kullet al., “Mixtures of Quaternary Ammonium Compounds and Long-chain FattyAcids as Antifungal Agents”, Applied Microbiology, 9:538-541 (1961). Thesynergism value (Q_(A)/Q_(a)+Q_(B)/Q_(b)) was determined. Q_(A) is theconcentration of potassium sorbate or sodium benzoate (in percent byweight) in the mixture, which yielded 100% retardation of the bacteria,i.e., resulted in a plate count of <10 cfu/g after 7 days. Q_(a) is theconcentration of potassium sorbate or sodium benzoate alone (in percentby weight) required to yield 100% retardation of the bacteria. Q_(B) isthe concentration of sodium erythorbate (in percent by weight) in themixture, which yielded 100% retardation of the bacteria. Q_(b) is theconcentration of sodium erythorbate alone (in percent by weight)required to yield 100% retardation of the bacteria.

[0101] When the value of (Q_(A)/Q_(a)+Q_(B)/Q_(b)) is less than one, themixture is synergistic. Values for (Q_(A)/Q_(a)+Q_(B)/Q_(b)) of 1 andgreater represent an additive effect and an antagonistic effect,respectively. The results are shown in Table 2 below. TABLE 2Q_(A)/Q_(a) + Preservative Mixture Q_(A) Q_(B) Q_(a) Q_(b) Q_(B)/Q_(b)75% potassium sorbate 0.225% 0.075% 0.45% 0.45% 0.67 and (<1) 25% sodiumerythorbate 75% Sodium Benzoate 0.225% 0.075% 0.45% 0.45% 0.67 and 25%Sodium erythorbate

Example 4

[0102] Each anionic shampoo sample in Table 3 below was tested asfollows. A standardized mixed bacterial solution was prepared accordingto the following procedure. 3 agar stabs of S. aureus (ATCC # 6538), P.aeruginosa (ATCC # 9027), and E. coli (ATCC # 8739) were separatelyincubated at about 35° C. for about 24 hours. Each stab was then washedwith 3 mL of sterile 0.85% saline solution. The washes of the 3 stabswere pooled together to form an organism mixture. The absorbance of theorganism mixture at 530 nm was adjusted to about 1.00 by adding saline.The spectrometer was calibrated with a saline blank. A 5 mL aliquot ofthe organism mixture was mixed together to produce the standardizedmixed bacterial solution. Then, 40 g of each shampoo sample wasinoculated with 0.2 mL of the standardized mixed bacterial solution andmixed. 1 g of the mixture was added to a sterile 20×150 mm screw captest tube.

[0103] 9 mL of sterile D/E neutralizer broth was added to the test tubeand mixed to form a 10⁻¹ dilution. Serial dilutions were preparedthrough to a 10⁻⁶ dilution with phosphate buffered water. The serialdilutions were plated onto Tryptic Soy Agar and incubated for 2 days atabout 35° C. Bacteria counts were performed after 0, 7, and 14 days. Theresults are shown in Table 1.

[0104] The anionic protein shampoo composition was comprised of 35% byweight of sodium lauryl ether sulfate; 25% by weight of triethanolaminelauryl sulfate; 3% by weight coconut diethanolamide (cocamide DEA); 1%by weight of hydrolyzed collagen, available as Polypro 5000™ from HormelFoods of Austin, Minn.; and 36% by weight of deionized water.

[0105] The cinnamaldehyde and other preservative containing samples wereprepared by mixing the appropriate amounts of the preservatives and theaforementioned anionic protein shampoo composition and heating themixture to about 50° C. for about 15 minutes. TABLE 3 S. aureus, P.aeruginosa, and E. coli (cfu/g) Shampoo Day 0 Day 7 Day 14 UnpreservedAnionic 3.0 × 10⁷ 3.0 × 10⁷ 3.0 × 10⁷ Protein Shampoo Composition 0.25%Cinnamaldehyde 3.0 × 10⁷ <10 <10 0.20% Cinnamaldehyde 3.0 × 10⁷ <10 <100.10% Cinnamaldehyde 3.0 × 10⁷ 1.0 × 10¹ <10 1.0% Benzyl Alcohol 3.0 ×10⁷ 5.0 × 10⁶ 5.3 × 10⁶ 1.0% LiquaPar Optima* 3.0 × 10⁷ 3.0 × 10⁷ 2.0 ×10⁷ 1% Tea Tree Oil 3.0 × 10⁷ 3.0 × 10⁷ 3.0 × 10⁷ 1% d-Limonene 3.0 ×10⁷ 3.0 × 10⁷ 3.0 × 10⁷ 1% Gerniol 3.0 × 10⁷ 3.0 × 10⁷ 3.0 × 10⁷ 1%Nerol 3.0 × 10⁷ 3.0 × 10⁷ 3.0 × 10⁷ 1% Citral 3.0 × 10⁷ 3.0 × 10⁷ 3.0 ×10⁷ 1% Eugenol 3.0 × 10⁷ 3.0 × 10⁷ 3.0 × 10⁷ 1% Hexahop 3.0 × 10⁷ 3.0 ×10⁷ 3.0 × 10⁷

[0106] All percentages in Table 3 are in percent by weight based upon100% by weight of total shampoo.

Example 5

[0107] Each anionic shampoo sample in Table 4 below was tested asfollows. A standard mixed bacterial solution was prepared according tothe following procedure. 2 agar slants of Candida albicans and 4 agarslants of Aspergillus niger were separately incubated at about 25° C.for about 48 hours and 7 days, respectively. Each slant was washed with3 mL of sterile 0.85% saline solution, collected and macerated in atissue grinder. Sufficient amounts of 0.85% saline solution were addedto each slant to obtain a visual count under a microscope with aNeubauer Hemocytometer of each innoculum of C. albicans and A. niger.Equal volumes of each standardized innoculum of C. albicans and A. nigerwere mixed together to form the standardized mixed fungal solution.

[0108] 40 g of each shampoo sample was inoculated with 0.4 mL of thestandardized mixed fungal solution and mixed. 1 g of the mixture wasadded to a sterile 20×150 mm screw cap test tube.

[0109] 9 mL of sterile D/E neutralizer broth was added to the test tubeand mixed to form a 10⁻¹ dilution. Serial dilutions were preparedthrough to a 10⁻⁶ dilution with phosphate buffered water. The serialdilutions were plated onto Sabourand dextrose agar and incubated 5 daysat about 25° C. Fungal counts were performed after 0 and 14 days. Theresults are shown in Table 9.

[0110] The anionic protein shampoo composition is described in Example4. The shampoo samples were prepared by mixing the appropriate amountsof the preservatives and the anionic protein shampoo composition andheating the mixture to about 50° C. for about 15 minutes. TABLE 4 FungalPlate Count (cfu/g) Shampoo Day 0 Day 7 Day 14 Unpreserved AnionicProtein 1.0 × 10⁵ 4.5 × 10⁴ 8.5 × 10⁴ Shampoo Composition 0.20Cinnamaldehyde 1.0 × 10⁵ <10 <10 0.10% Cinnamaldehyde 1.0 × 10⁵ 3.0 ×10¹ <10 0.05% Cinnamaldehyde 1.0 × 10⁵ 8.0 × 10³ <10 1.0% Benzyl Alcohol1.0 × 10⁵ 6.0 × 10³ 6.0 × 10⁴ 1.0% LiquaPar Optima 1.0 × 10⁵ 4.0 × 10⁴3.0 × 10⁴

Example 6

[0111] Each cream sample in Table 5 below was tested by the proceduredescribed in Example 1. A glyceryl monostearate (GMS) cream as describedin Table 3 below was prepared as follows. The polyoxyethylene glycerylmono stearate, glyceryl mono stearate, cetearyl alcohol, and myristylpropionate were mixed and heated to 60° C. in a first container. Theglycerin and sterile deionized water were mixed and heated to 60° C. ina second container. The solution in the first container was poured intothe second container. The second container was maintained at 60° C. for10 minutes. The solution in the second container was allowed to cool.The pH of the solution was adjusted to pH 7 with sodium hydroxide toyield the GMS cream. TABLE 5 Ingredient Trade Name Chemical Name Amount(% w/w) Aldosperse ® MS-20 Polyoxyethylene (POE) 4.00 (Lonza) glycerylmonostearate Aldo ® (Lonza) Glyceryl monostearate 6.00 TA 1618 (Proctor& Cetearyl alcohol 1.50 Gamble) Lonzest ® 143-S (Lonza) Myristylpropionate 8.00 Glycon ® G-100 (Lonza) Glycerin 5.00 — Sterile DeionizedWater 75.50 Total 100.00

[0112] The cream samples shown in Table 6 below were prepared by mixingthe appropriate amounts of the preservatives and the GMS cream andheating the mixture to 50° C. for 10-15 minutes. The results are shownin Table 6 below. TABLE 6 S. aureus, P. aeruginosa, and E. coli (cfu/g)Cream Day 0 Day 7 Day 14 Unpreserved GMS Cream 3.0 × 10⁷ 3.0 × 10⁷ 3.0 ×10⁷ 0.25% Cinnamaldehyde 3.0 × 10⁷ <10 <10 0.10% Cinnamaldehyde 3.0 ×10⁷ 4.0 × 10⁴ 9.3 × 10⁵

[0113] The cream samples shown in Table 7 below were prepared by mixingthe appropriate amounts of the preservatives and the GMS cream andheating the mixture to 50° C. for 10-15 minutes. The results are shownin Table 7 below. TABLE 7 Fungal Plate Count (cfu/g) Cream Day 0 Day 7Day 14 Unpreserved GMS Cream 1.0 × 10⁵ 2.3 × 10⁵ 1.5 × 10⁵ 0.25%Cinnamaldehyde 1.0 × 10⁵ <10 <10 0.10% Cinnamaldehyde 1.0 × 10⁵ <10 <10

Example 7

[0114] The procedure in Example 4 was repeated with the shampoo samplesshown in Table 8 below. The results are shown in Table 8. TABLE 8 S.aureus, P. aeruginosa, and E. coli (cfu/g) Cream Day 0 Day 7 Day 14Unpreserved Anionic Protein 3.0 × 10⁶ 3.0 × 10⁷ 3.0 × 10⁷ shampooComposition 0.10% Cinnamaldehyde 3.0 × 10⁶ 1.0 × 10¹ <10 0.05%Cinnamaldehyde 3.0 × 10⁶ 6.5 × 10⁶ 1.0 × 10⁷ 0.05% Glydant 2000 ™* 3.0 ×10⁶ 2.0 × 10² 1.0 × 10² 0.02% Glydant 2000 ™ and 3.0 × 10⁶ <10 <100.025% Cinnamaldehyde

[0115] Synergism for the cinnamaldehyde/Glydant 2000™ solutions in Table8 against S. aureus, P. aeruginosa, and E. coli was calculated by themethod described in C. E. Kull et al., “Mixtures of Quaternary AmmoniumCompounds and Long-chain Fatty Acids as Antifungal Agents”, AppliedMicrobiology, 9:538-541 (1961). The synergism value(Q_(A)/Q_(a)+Q_(B)/Q_(b)) in Table 7 was determined. Q_(A) is theconcentration of cinnamaldehyde (in percent by weight) in the mixture,which yielded 100% retardation of the bacteria, i.e., resulted in aplate count of <10 cfu/g after 14 days. Q_(a) is the concentration ofcinnamaldehyde alone (in percent by weight) required to yield 100%retardation of the bacteria. Q_(B) is the concentration of Glydant 200™(in percent by weight) in the mixture, which yielded 100% retardation ofthe bacteria. Q_(b) is the concentration of Glydant 2000™ alone (inpercent by weight) required to yield 100% retardation of the bacteria.

[0116] When the value of (Q_(A)/Q_(a)+Q_(B)/Q_(b)) is less than one, themixture is synergistic. Values for (Q_(A)/Q_(a)+Q_(B)/Q_(b)) of 1 andgreater than 1, represent an additive effect and an antagonistic effect,respectively.

[0117] The results are shown in Tables 9 and 10 below. TABLE 9 For Day 7Q_(A)/Q_(a) + Preservative Mixture Q_(A) Q_(B) Q_(a) Q_(b) Q_(B)/Q_(b) 0.05% Glydant 2000 ™ — — 0.05% — —  0.10% Cinnamaldehyde — — — 0.1% — 0.02% Glydant 2000 ™ 0.02% 0.025% — — 0.65 and 0.025% Cinnamaldehyde

[0118] TABLE 10 For Day 14 Q_(A)/Q_(a) + Preservative Mixture Q_(A)Q_(B) Q_(a) Q_(b) Q_(B)/Q_(b)  0.05% Glydant 2000 ™ — — 0.05% — —  0.10%Cinnamaldehyde — — — 0.05% —  0.02% Glydant 2000 ™ 0.02% 0.025% — — 0.90and 0.025% Cinnamaldehyde

Example 8

[0119] The Minimum Inhibitory Concentration (MIC) of the preservativemixtures was tested. The MIC is the lowest concentration of aningredient that will inhibit the growth of an organism. This study wasconducted using the Hamilton Micro Lab AT Plus Autodilutor Liquidhandling System. The programs for the auto-dilutor were based on Lonza'sStandard Application Method SAPM# 412-01-1. The Hamilton Autodilutor wasused to dilute the starting concentrations of the preservativecombination by 50% using nutrient broth in 96 well micro titer platesand also to inoculate the microorganism in the test samples.

[0120] This preservatives tested were Isocil™ (a blend of methylisothiazolinone and methyl-chloro-isothiazolinone), Benzocil™(benzisothiazolinone) and Lonzagard™ (benzethonium chloride), all ofwhich are available from Lonza Inc. of Fair Lawn, N.J., in variousconcentrations and combinations with cinnamaldehyde. Controls were alsoincluded in each test plate. Each preservative combination was tested induplicate against Staphylococcus aureus (ATCC # 6538) and Escherichiacoli (ATCC # 8739).

[0121] Test plates were diluted by the Hamilton Autodilutor and theninoculated with the test organism to achieve approximately 10⁶ colonyforming units/gram in the test sample (cfu/g). The plates were thenincubated in a 32 degree Celsius oven for 72 hours. Results weredetermined by checking for growth in the test samples versus the controlwells on each plate (visual determination of turbidity in the wells).The MIC shown below in Table 11 was reported as the lowest test levelsof preservative or preservative blend that did not show any growth.

[0122] A mixture of 7.5 ppm (active) Benzocil™ and 25 ppm cinnamaldehydeeffectively inhibited growth. Also, a mixture of 0.47 ppm (active)Isocil™ and 6.3 ppm cinnamaldehyde effectively inhibited growth. TABLE11 Test Material MIC for S. Aureus Cinnamaldehyde   125 ppm Isocil ™(Isothiazolinone) 0.585 ppm (active) Benzocil ™ (Benzoisothiazolinone)9.375 ppm (active)

[0123] Synergism values for the Isocil™/cinnamaldehyde andBenzocil™/cinnamaldehyde combinations were calculated from the MICvalues reported in Table 11 by the method described in Kull, supra,referred to above, and are set forth in Tables 12 and 13, below. TABLE12 Q_(A)/Q_(a) + Preservative Mixture Q_(A) Q_(B) Q_(a) Q_(b)Q_(B)/Q_(b) 0.585 ppm (active) Isocil ™ — — 0.585 — —   125 ppmCinnamaldehyde — — — 125 —  0.47 ppm (active) Isocil ™ 0.47 6.3 — — 0.85and 6.3 ppm Cinnamaldehyde

[0124] TABLE 13 Q_(A)/Q_(a) + Preservative Mixture Q_(A) Q_(B) Q_(a)Q_(b) Q_(B)/Q_(b) 9.375 ppm (active) — — >9.375 — — Benzocil ™   125 ppmCinnamaldehyde — — — >125 —  7.5 ppm (active) Benzocil ™ 75 25 — — <1.0and 25 ppm Cinnamaldehyde

Example 9

[0125] The color stability of the cinnamaldehyde/potassium sorbatemixtures described below were tested with a Gardner color test.Hydrochloric acid was added to adjust the pH of the formulation to thepH specified. The results are shown below None Hydrochloric AcidTemperature Room Room Stabilizer Initial Temperature 37° C. Temperature37° C. Initial pH 10.60 10.60 10.60 8.96 8.96 Color 6 10-11 14-15 7-811-12 Water 33.9 34.2 34.1 34.0 34.4 K sorbate 41.5 42.3 42.3 44.0 43.4Cinnamaldehyde 14.8 15.1 14.9 15.5 15.6 Final pH 9.91 9.83 9.82 8.678.67

[0126] All patents, applications, articles, publications, and testmethods mentioned above are hereby incorporated by reference.

[0127] Many variations of the present invention will suggest themselvesto those skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

We claim:
 1. An antimicrobial composition comprising an antimicrobialeffective amount of a mixture comprising at least two of: (a) lemongrass oil; (b) cinnamaldehyde, cinnamon oil, cinnamomum cassia, cinnamonextract, cassia leaf oil, 3,4-dihydroxycinnamic acid or salt thereof, ora mixture thereof; (c) sorbic acid, or a salt thereof; (d) erythorbicacid, or a salt thereof; (e) benzoic acid, or a salt thereof; (f)arabinogalactan, galactoarabinan, or a mixture thereof; (g) ahexahydro-iso-alpha-acid, tetrahydro-iso-alpha-acid, or a mixturethereof; (h) Achillea fragrantissima oils, Santolina fragrantissimaoils, Forssk oils, Lavender cotton oils; and (i) Glucono Delta Lactone.2. The antimicrobial composition of claim 1, wherein the mixturecomprises (a) cinnamaldehyde; and (b) sorbic acid or a salt thereof. 3.The antimicrobial composition of claim 2, wherein component (b) the saltis potassium sorbate.
 4. The antimicrobial composition of claim 1,wherein the mixture comprises (a) erythorbic acid or a salt thereof, and(b) one or more of citric acid, glucono delta lactone, benzoic acid,sorbic acid, EDTA, or a salt thereof.
 5. The antimicrobial compositionof claim 1, wherein the mixture comprises (a) benzoic acid or a saltthereof, and (b) one or more of citric acid, glucono delta lactone,sorbic acid, EDTA, or a slat thereof.
 6. The antimicrobial compositionof claim 4, wherein the mixture comprises potassium sodium erythorbateand potassium sorbate.
 7. The antimicrobial composition of claim 6,further comprising benzoic acid or a salt thereof.
 8. The antimicrobialcomposition of claim 7, wherein the salt of benzoic acid is sodiumbenzoate.
 9. The antimicrobial composition of claim 5, wherein themixture comprises (a) sodium benzoate and sodium erythorbate.
 10. Theantimicrobial composition of claim 1, further comprising a solvent. 11.The antimicrobial composition of claim 10, wherein the solvent isselected from water, glycols, alcohols, and mixtures thereof.
 12. Theantimicrobial composition of claim 11, wherein the solvent is a mixtureof water and a glycol.
 13. The antimicrobial composition of claim 12,wherein the glycol is glycerin.
 14. The antimicrobial composition ofclaim 10, wherein the solvent is a mixture of water and an alcohol. 15.The antimicrobial composition of claim 14, wherein the alcohol isethanol.
 16. The antimicrobial composition of claim 1, wherein themixture comprises (a) cinnamaldehyde and (b) sorbic acid or a saltthereof.
 17. The antimicrobial composition of claim 1, wherein thecomposition mixture is present at a concentration of from about 0.01 toabout 2% by weight, based on 100% weight of the total composition.
 18. Amethod of killing and/or inhibiting the growth of microorganisms on asubstrate comprising applying an effective amount of the antimicrobialcomposition of claim 1 to the substrate.
 19. The method of claim 18,wherein the microorganisms are selected from S. aureus, P. aeruginosa,E. coli, Candida albicans, Aspergillus niger and Phytophthora ramrum.20. A product other than a foodstuff, pharmaceutical, or cosmeticcomprising a preservative effective amount of cinnamaldehyde, whereinthe product is substantially free of parabens.
 21. The product of claim20, wherein the product is a household, industrial, or institutionalproduct.
 22. The product of claim 20, wherein the product is a personalcare product.
 23. The product of claim 22, wherein the personal careproduct is a shampoo, body lotion, conditioner, or soap.
 24. The productof claim 20, wherein the product comprises less than a smellingeffective amount of cinnamaldehyde.
 25. The product of claim 20, whereinthe product is free of cinnamon oil.
 26. A preservative formulationcomprising an antimicrobial effective amount of a synergistic mixturecomprising (a) cinnamaldehyde; and (b) at least one alkanol substituteddialkylhydantoin.
 27. The preservative formulation of claim 26, whereinthe wherein the alkanol substituted dialkyl hydantoin has the formula:

wherein R₁ and R₂ are each independently hydrogen or (CH₂)OH, with theproviso that both R₁ and R₂ cannot be hydrogen, and R₃ and R₄ are eachindependently methyl, ethyl, propyl, or aryl.
 28. The preservativeformulation of claim 27, wherein the alkanol substituteddialkylhydantoin is dimethylol dimethylhydantoin.
 29. A method ofkilling and/or inhibiting the growth of microorganisms on a productother than a foodstuff, pharmaceutical, or cosmetic, the methodcomprising applying an effective amount of cinnamaldehyde to theproduct, wherein the product is free of parabens.
 30. A method ofinhibiting the growth of microorganisms comprising applying an effectiveamount of the preservative formulation of claim
 26. 31. A method ofkilling and/or inhibiting the growth of fungi on a substrate comprisingapplying an effective amount of cinnamaldehyde to the product, whereinthe product is free of parabens.
 32. A method of inhibiting the growthof fungi on a substrate comprising applying an effective amount of thepreservative formulation of claim 26.